Glucocorticoid-mediated induction of ZBTB16 affects insulin secretion in human islets and EndoC-βH1 β-cells

Summary Glucocorticoid use is associated with steroid-induced diabetes mellitus and impaired pancreatic β-cell insulin secretion. Here, the glucocorticoid-mediated transcriptomic changes in human pancreatic islets and the human insulin-secreting EndoC-βH1 cells were investigated to uncover genes involved in β-cell steroid stress-response processes. Bioinformatics analysis revealed glucocorticoids to exert their effects mainly on enhancer genomic regions in collaboration with auxiliary transcription factor families including AP-1, ETS/TEAD, and FOX. Remarkably, we identified the transcription factor ZBTB16 as a highly confident direct glucocorticoid target. Glucocorticoid-mediated induction of ZBTB16 was time- and dose-dependent. Manipulation of ZBTB16 expression in EndoC-βH1 cells combined with dexamethasone treatment demonstrated its protective role against glucocorticoid-induced reduction of insulin secretion and mitochondrial function impairment. In conclusion, we determine the molecular impact of glucocorticoids on human islets and insulin-secreting cells and investigate the effects of glucocorticoid targets on β-cell function. Our findings can pave the way for therapies against steroid-induced diabetes mellitus.

Glucocorticoids (GCs) alter the transcriptome of human islets and EndoC-bH1 cells ZBTB16 is a highly confident direct GC target in human islets and EndoC cells GCs negatively affect insulin secretion and strongly induce ZBTB16 in EndoC cells

INTRODUCTION
Glucocorticoids are steroid hormones secreted by the adrenal glands and mediate diverse immunological and metabolic effects in vertebrates. 1 Among these, their anti-inflammatory, anti-allergic, and immunomodulatory properties are the main reason glucocorticoid analogs, such as dexamethasone, are widely prescribed in everyday clinical settings. 2,3 Inside cells glucocorticoids act as ligands that bind to the cytosolic glucocorticoid receptor (GR), a member of the soluble nuclear receptor superfamily of ligand-dependent transcription factors, 4 which is then activated and translocated into the nucleus. Once in the nucleus, GR binds to specific genomic elements in the vicinity of its target genes called glucocorticoid responsive elements (GREs), leading to transcriptional induction or repression of these genes. 5,6 While GR is ubiquitously expressed in all tissues and GREs have been identified in various cell types, 7 the glucocorticoid-responsive gene sets are cell-type specific with modest overlap between cells. 8 Although endogenous glucocorticoids are essential in maintaining glucose homeostasis, the use of exogenous, more potent glucocorticoids used in the clinical setting has been associated with adverse metabolic effects, such as hyperglycemia and steroid-induced diabetes mellitus. 9,10 Although steroid-induced diabetes mellitus has been mainly attributed to glucocorticoid-induced whole-body insulin resistance, 11 growing evidence also suggest a deleterious effect of glucocorticoids on pancreatic b-cells. In vitro and in vivo studies in rodents [12][13][14] and humans 15,16 have demonstrated impaired b-cell function and/or reduced glucose-stimulated insulin secretion after acute and chronic glucocorticoid treatment. The consequences are more severe in susceptible individuals with reduced insulin sensitivity 17 or glucose-stimulated insulin secretion 18 before glucocorticoid treatment, as well as in obese females, 19 first-degree relatives of type 2 diabetes patients 20 and those at a higher age. 21 It is worth mentioning that endogenous glucocorticoids such as cortisone and cortisol do not affect b-cell function and insulin secretion under physiological concentrations. 22 In a recent study dexamethasone and glucolipotoxicity showed a synergistic negative effect on insulin secretion in EndoC-bH1 cells partly explained by an increased proinsulin/insulin ratio. 23 Glucocorticoidinduced impairment of glucose-stimulated insulin secretion has been linked to reduced expression of genes important for b-cell function including transcription factors such as PDX-1 and NKX6-1, the exocytotic protein SYT13, and the glucocorticoid receptor GR 16 and increased expression of glucocorticoidregulated kinase 1 (SGK1). 16,24 Upregulation of SGK1 increases the activity of voltage-gated K + channels, which in turn reduces Ca 2+ entry into the b-cell and thereby reduces glucose-stimulated insulin secretion. 24 We recently showed that regulation of these proteins by glucocorticoids is in an intricate interplay with the lincRNA GAS5. 16 ZBTB16 (zinc finger and BTB domain containing 16) is a known glucocorticoid target in non-pancreatic cell types. [25][26][27] It has been shown to be involved in distinct biological processes such as self-renewal and differentiation of various stem cell types, 28 limb development, 29 spermatogenesis maintenance, 30 and hematopoiesis. 31 Despite being very lowly expressed in the endocrine cells of human islets, 32 substantial induction of ZBTB16 has recently been described in human islets after glucocorticoid treatment. 33 However, the exact function of ZBTB16 in pancreatic islets is still not known.
Despite the characterization of important glucocorticoid gene targets in the islets and b-cells, we still have limited knowledge on the pathways regulated by glucocorticoids that lead to impaired insulin secretion. Thus, we performed differential gene expression analysis using RNA-sequencing (RNA-seq) data on both human pancreatic islets and the human b-cell line EndoC-bH1 after treatment with dexamethasone, a synthetic glucocorticoid widely used in the clinical setting. Furthermore, we integrated publicly available chromatin immunoprecipitation sequencing (ChIP-seq), chromatin state, and human enhancer data to rank our RNA-seq-defined gene targets according to their potential of being direct targets of GR. Finally, we performed functional validation and gene target identification on the top-ranked gene ZBTB16.

RESULTS
Human islet and EndoC-bH1 cells display extensive transcriptomic changes after high-dose dexamethasone treatment We first determined transcriptome-wide changes due to dexamethasone treatment ( Figure 1A) in human islets and the human insulin-secreting cell line EndoC-bH1. EndoC-bH1 cells are widely used as  iScience Article translational human b-cell models due to the substantial global omics overlap with primary adult human bcells, including their transcriptome, proteome, and secretome. 34 Dexamethasone can affect hundreds of target genes and previous work in human islets and insulin-secreting cells have focused on key proteins in glucocorticoid signaling and b-cell function. 16,23,33 Human islets (characteristics in Table S1) and En-doC-bH1 cells were treated for 48h and 24h, respectively, in the absence and presence of 2 mM dexamethasone before transcriptomic characterization. Analysis of the transcriptomic profiles of human islets and EndoC-bH1 cells identified 1473 and 3147 differentially expressed (DE) genes (adjusted p-value < 0.05), respectively ( Figure 1A and Table S2). The overlap of DE genes between the dexamethasone-treated human islets and EndoC-bH1 cells was 581 genes ( Figure 1B). Interestingly, the expression of these genes was altered in the same direction in all samples, with 309 genes being upregulated and 272 genes downregulated (Table S2). The consistency in the direction of the expression of the overlapping genes implies robust transcriptomic changes in the human b-cell and validates the results.
Functional annotation of the 581 shared DE genes revealed several enriched pathways important for b-cell function, such as glucose homeostasis, insulin processing, insulin secretion, regulation of b-cell gene expression, and b-cell proliferation and differentiation ( Figure 1C, full list in Table S3). The pathway insulin secretion consisted of important genes for b-cell identity such as NKX6-1 and PDX-1. When we instead performed functional annotation analysis of DE genes specific for either human islets (892 genes) or EndoC-bH1 cells (2566 genes) distinct genes were found to be involved in insulin secretion, glucose homeostasis, and post-translational processing. Pathways related to insulin secretion processes were more profoundly enriched in EndoC-bH1 cells. Moreover, the EndoC-bH1 DE gene set displayed a high enrichment of terms relevant to endoplasmic reticulum (ER) stress, intrinsic apoptotic signals, and cell cycle regulation, while the human islet DE gene set was enriched for terms associated with inflammation, extrinsic apoptotic pathways, response to insulin, insulin resistance, lipid metabolism, and glucocorticoid response ( Figure 1C).

Glucocorticoid Receptor regulates its targets in a distal manner and its action depends on auxiliary transcription factors
We next investigated the GR binding properties of genes DE by dexamethasone in human islets and En-doC-bH1 cells. Glucocorticoids exert their effects by binding to the GR which is internalized and acts as a transcription factor. First, we obtained a high-confidence comprehensive GR genomic binding site list derived from various chromatin immunoprecipitation sequencing (ChIP-seq) experiments from the GTRD database (GR-ChIP sites). 35 Next, we associated the obtained DE genes in islet and EndoC-bH1 sets ( Figure 1) with GR-ChIP sites within a 150 kb window of their transcription start site (TSS), to yield GREs in the DE genes. After that, we calculated the estimate of the likelihood of the gene being regulated by GR using a formula that considers the distance of every associated GRE, giving a higher score with a smaller distance. 36,37 We found that the potential of a gene to be regulated by GR was significantly higher for islet and EndoC-bH1 cell DE genes than non-DE genes, indicating that more GREs are detected closer to upregulated or downregulated DE genes than non-DE genes ( Figure 2A). Moreover, the vast majority (z95%) of GREs that were associated with the DE genes were located further from 3 kb of their TSS in both islets and EndoC-bH1 cells, suggesting that GR regulation is carried out in a distal manner ( Figure 2B).
De novo motif discovery in the DNA sequences of all GREs revealed 4 enriched motifs that correspond to DNA binding sequences of distinct transcription factor families ( Figure 2C). In addition to a motif that coincides with the GR binding sequence (GBS), motifs belonging to the AP-1, ETS/TEAD, and FOX transcription factor families were found to be enriched. Surprisingly, when GBS was scanned across GREs, only 37% of GREs displayed a stringent GBS (p < 0.0001), while 77% contained a less canonical GBS (p < 0.001). A very high proportion of GREs (95%) contained at least one of the alternative AP-1, ETS/TEAD, and FOX binding sequences besides a GBS, with the proportion rising to 97% for GREs that did not contain any canonical GBS.
ZBTB16 is the most strongly predicted direct glucocorticoid target in human islets and EndoC-bH1 cells We next developed a bioinformatics pipeline ( Figure 3A, described in detail in STAR Methods), to focus on potential direct glucocorticoid targets from the large list of DE genes after high-dose dexamethasone treatment. Briefly, we first associated each DE gene with GR-ChIP sites to generate GREs. Then by integrating human islet/EndoC-bH1-specific accessible chromatin region data, enhancer-target association data, and GR binding motif site information, we introduced a Normalized Annotation Score. iScience Article the Normalized Annotation Score with the fold-change values of each DE gene, a gene rank product was generated, which can be interpreted as a p-value that indicates the potential of the gene to be a direct glucocorticoid target. In both human islet and EndoC-bH1 cell sets, ZBTB16 had the lowest rank product, making it the most suitable candidate to be a direct target of dexamethasone and, consequently, by GR ( Figure 3B). Indeed, ZBTB16 transcriptional induction was among the highest in human islets and En-doC-bH1 cells ( Figure 3C), and both RNA and protein levels of ZBTB16 were strongly induced upon high-dose dexamethasone treatment in EndoC-bH1 cells ( Figure 3D).
Glucocorticoid Responsive Elements are located intronically and within conserved regions of ZBTB16 in EndoC-bH1 cells  iScience Article (Positions: 5, 6, 8, and 10; positions 8 and 10 contain more than one GBS), all of which are located in highly conserved regions. Taken together, we hypothesized that the induction of ZBTB16 expression is possibly related to the differential binding of GR to one or several of the ZBTB16 identified GREs. To prove our hypothesis, we performed ChIP followed by PCR in EndoC-bH1 cells pre-treated in the absence or presence of 100 nM dexamethasone for 24h. GR binding was validated in all the 10 ZBTB16 GREs ( Figure 4C) and we observed a consistent active binding in the treated samples compared to the non-treated samples, although with some degree of variability ( Figure S1). The presence of multiple GREs within ZBTB16 suggests a highly dynamic transcriptional regulation of this gene resulting in its hyperactivation upon glucocorticoid treatment.

Dexamethasone induces ZBTB16 expression in a dose-and time-dependent manner and impairs insulin secretion in EndoC-bH1 cells
To further investigate the conditions under which ZBTB16 transcription is induced in the b-cells, we measured the expression levels of ZBTB16 after treatment with different doses of dexamethasone (0.1-2000 nM) for 24h in EndoC-bH1 cells ( Figure 5A). The ZBTB16 expression at the different concentrations of dexamethasone was compared with those of genes that are known to be involved in the iScience Article glucocorticoid-GR signaling pathway in b-cells, such as SGK1 (activated) and GR (repressed). 16 As shown in Figure 5A, there were significant differences in the expression of the genes that depended on glucocorticoid dose (two-way ANOVA: gene expression*dose p < 0.001). The pairwise comparisons showed an increasing expression of ZBTB16 in response to dexamethasone concentration in the range of 10-1000 nM, which is higher than that of SGK1. Since 100 nM appeared to be the lowest dexamethasone concentration in which we observed significant target gene induction, we used this concentration as our working concentration in subsequent functional experiments. At the same time, treatment with increasing dexamethasone concentrations also led to a significantly elevated proliferation rate ( Figure 5B).
We next measured the expression of the same genes in EndoC-bH1 cells after incubation with or without 100 nM dexamethasone for an increasing time period (2h, 8h, 24h, and 48h; Figure 5C). The results demonstrated significant differences in the expression of genes that depend on time under treatment (two-way ANOVA: gene expression*time p < 0.05, Figure 5C). Pairwise comparisons reveal that ZBTB16 expression is significantly induced 48h post-treatment (compared to 2h post-treatment) and it is significantly more expressed than SGK1 24h and 48h post-treatment.
We also asked whether the treatment with 100 nM dexamethasone has a time-dependent effect on glucose-stimulated insulin secretion. For that reason, we performed insulin secretion assay in the presence of 1-and 20-mM glucose in EndoC-bH1 cells after incubation with 100 nM dexamethasone at different time points (2h, 8h, 24h, and 48h). Analysis showed that insulin secretion was significantly different depending on the glucose concentration and incubation time (3-way ANOVA: insulin secretion*glucose concentration*time p < 0.001, Figure 5D). Pairwise comparisons showed that glucose-stimulated insulin secretion is significantly reduced after a 48h dexamethasone treatment period. Dexamethasone caused a significant reduction of the cellular insulin content across time under treatment (3-way ANOVA: insulin content*time p < 0.05), which is apparent and consistent 8h post-treatment ( Figure 5E). This can partly explain the reduced insulin secretion that was observed.
Induction of ZBTB16 expression can be protective for the b-cell We were next interested in whethter increased levels of ZBTB16 are partly responsible for the deleterious negative effects of glucocorticoid treatment on insulin secretion. We, therefore overexpressed ZBTB16 in EndoC-bH1  Figures 6A-B). Cells were then incubated in the absence or presence of 100 nM dexamethasone for 48h before insulin secretion assay is performed. Overexpression of ZBTB16 affected glucose-stimulated insulin secretion independently of the dexamethasone treatment (3-way ANOVA: ZBTB16 overexpression * dexamethasone treatment * glucose concentration p = 0.13, ZBTB16 overexpression p = 0.006) by slightly rescuing the dexamethasone-induced impairment of insulin secretion ( Figure 6C-left). This effect was accompanied by increasing cellular insulin content ( Figure 6C-right).
We investigated whether the induction of ZBTB16 could be involved in gene regulatory programs mediated by the glucocorticoid-GR regulatory axis in b-cells, by determining the gene expression of the glucocorticoid gene targets SGK1, GR, PDX-1, as well as the insulin gene (INS). ZBTB16 induction did not alter the expression of any of those genes ( Figures 6D-G), suggesting that ZBTB16 is not mediating the glucocorticoid-induced expression changes of SGK1 or PDX-1.
Next, we attempted to suppress ZBTB16 induction after dexamethasone treatment and measure glucosestimulated insulin secretion and expression of the glucocorticoid gene targets. Knockdown of ZBTB16 under this condition led to a partial ZBTB16 suppression of induction by z 30% (Figures 6H-I). We found that the partial reduction in ZBTB16 expression influenced insulin secretion independently of the dexamethasone treatment and glucose concentration (3-way ANOVA: ZBTB16 suppression * dexamethasone iScience Article treatment * glucose concentration p = 0.2, ZBTB16 suppression p = 0.002) and it led to increased insulin secretion under low glucose treatment both in the control and the treated samples ( Figure 6J-left). Insulin content was found to be reduced in the ZBTB16-suppressed samples compared to the control ( Figure 6Jright). Partial suppression also caused changes in the glucocorticoid-mediated gene expression patterns, as SGK1 induction was inhibited ( Figure 6K). However, expression of GR and PDX-1 were unaltered under the same condition ( Figure 6L-M). Insulin transcription was reduced in the treated cells with partially suppressed ZBTB16 (Figure 6N), which is in line with the observed reduction of insulin content.
Furthermore, to evaluate the cellular function of ZBTB16 we used our bioinformatics pipeline to identify potential direct gene targets of ZBTB16 in human islets ( Figure 7A and STAR Methods). The analysis  iScience Article detected 1093 predicted gene targets (Table S4 -tab A). By analyzing the transcriptome of human islet b-cells derived from a published single-cell study (GEO: GSE153855) we found that 751 of these targets are also expressed in the human b-cells ( Figure 7B and Table S4 -tab B). Functional annotation of the ZBTB16 target genes expressed in either human islets or b-cells revealed similar enriched terms/pathways related to rRNA and mRNA processing, ER protein targeting, cell cycle regulation, and mitochondrial membrane organization among others ( Figure 7C, full lists in Table S5). Thus, we next sought to get a deeper understanding of the mechanism of action of ZBTB16 by investigating cell proliferation and mitochondrial function in EndoC-bH1 cells under dexamethasone treatment after ZBTB16 manipulation. As mentioned before, the proliferation rate after dexamethasone treatment is considerably increased (Figure 5B) and ZBTB16 induction or suppression did not seem to have any additional effects ( Figure S3).
Regarding mitochondrial function, we performed mitochondrial oxygen consumption rate (OCR) measurements in EndoC-bH1 cells, which were assessed as whole cell OCR after overexpressing ZBTB16 and treating with dexamethasone. The overall effect of dexamethasone was a clear suppression of mitochondrial respiration, indicating impaired mitochondrial function, as outlined by a general lowering of OCR during pyruvate and carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) measurements across the entire recording trace ( Figure 7D). This suppression was no longer significant in EndoC-bH1 cells overexpressing ZBTB16 under dexamethasone treatment, indicating that overexpression of ZBTB16 rescues mitochondrial function. Basal respiration was found to be higher in cells overexpressing ZBTB16 under dexamethasone treatment than those in control cells exposed or not in dexamethasone ( Figure 7E). Acute response measured as increase in OCR upon pyruvate injection, ATP production assessed as decrease in OCR after oligomycin injection that inhibits ATP-synthase, and maximal respiration as measured after the addition of an uncoupler of the inner mitochondrial membrane (FCCP), all showed the same trend where dexamethasone treatment consistently led to significantly reduced magnitude of OCR responses to these injections ( Figures 7F-H). Overexpression of ZBTB16, however, did not show the same degree iScience Article of reduction, once again showing that ZBTB16 can rescue these aspects of mitochondrial function.
Coupling efficiency was not altered by dexamethasone treatment or by overexpressing ZBTB16 (Figure 7I).

DISCUSSION
Multiple studies have highlighted the contribution of the pharmacological use of glucocorticoids as a mediator of T2D development, including impaired insulin secretion. However, a comprehensive understanding on how glucocorticoids affect b-cell function has not been fully reached. In this study, we performed RNA-seq analysis that allowed us to compare transcriptomic changes after glucocorticoid treatment in human pancreatic islets and the human insulin-secreting cell line EndoC-bH1. Integration of our transcriptomic results with ChIP-seq GR binding, open chromatin state, and enhancer data from publicly available resources allowed us to study GR binding properties in both human islets and EndoC-bH1 cells and identify ZBTB16 as a highly confident direct gene target of GR. Finally, we functionally verified that ZBTB16 is 1) strongly induced in EndoC-bH1 cells and 2) most likely has a protective role by increasing insulin secretion, limiting the otherwise deleterious reduction of glucose-stimulated insulin secretion by glucocorticoids.
Dexamethasone was selected as the synthetic glucocorticoid to perform all in vitro experiments in this study. This was because several studies that were focused on the glucocorticoid effect in human islets or human b-cell lines 16,23,33 or the glucocorticoid genomic response in other human cell lines [38][39][40] also utilized dexamethasone and, in that way, we would be able to directly compare, evaluate, and discuss the conclusions of those studies more accurately. Interestingly, approximately 40% of the DE genes in human islets after dexamethasone treatment were differentially expressed in the human b-cell line EndoC-bH1. Functional annotation analysis revealed these genes to be involved in pathways crucial for b-cell function and development. Many of these pathways are already known to be implicated in glucocorticoid-mediated impaired insulin secretion in rodent islets or cell lines. These include glucose metabolism, 41 increased potassium channel activity, 24 calcium transport and insulin secretion, 12,13,41 b-cell proliferation, 42,43 and insulin processing. 44 Other pathways such as pancreas development, differentiation and lipid metabolic process, and localization have not been previously studied in a b-cell context to the best of our knowledge. Hence, the effect on gene expression between human islets and EndoC-bH1 cells has many similarities. Moreover, among the distinctly DE genes between islets and EndoC-bH1 cells several of them are involved in identical pathways related to glucose metabolism, insulin secretion, and translational modifications, while other genes in unrelated pathways. Human islet-specific glucocorticoid-induced changes, such as genes related to insulin response, may be associated with the existence of other pancreatic cell types in the islets, such as the a-cells, as glucagon secretion has been found to be reduced in mouse islets after acute dexamethasone treatment. 45 The molecular mechanisms regulating glucocorticoid-induced changes in gene expression are complex and can differ between cell types. 46,47 Here, by using ChIP-seq data we showed that the majority of GRE sites are located further than 3 kb away from the TSS of the associated genes, confirming that GR is not characterized by the typical promoter-proximal binding pattern. 39,48 Furthermore, a significantly higher number of GREs were detected closer to upregulated or downregulated DE genes than non-DE genes, supporting a role of GR as both an activator and repressor of gene expression. 49,50 It was also apparent from our data that GR binding was more strongly associated with the induced rather than the repressed DE genes, which is consistent with results from GR global recruitment experiments in other human cell lines. 39,51 Although this may imply that the mechanism underlying GR repression depends on long-range interactions between a smaller number of more distal GREs and the TSS of target genes, 39 other studies suggest that the lower GRE abundance near downregulated DE targets may be due to the GR binding to distinct negative GREs (nGREs) with low-affinity, 52 which are untraceable by ChIP-seq peak calling methods because of their weak ChIP-seq signal. 53 To get a deeper understanding of the Glucocorticoid Receptor Binding Sequence (GBS) within the GREs near the glucocorticoid-regulated DE genes in islets and EndoC-bH1, we performed a de novo motif discovery. Only a proportion (z77%) of GREs seems to encompass the standard 15-bp GBS, 54 consistent with previous reports indicating a similar proportion (60-80%) on different human cell lines. 8,39,55 This could explain why a small fraction of classical GBSs in the genome is actually occupied by GR. 48 Our data also demonstrates the important role of additional TFs in GR gene regulation. The vast majority of the GREs in our data (95%) were found to be composite response elements containing at least one alternative motif ll OPEN ACCESS iScience 26, 106555,  iScience Article beside the GBS, indicating that a big part of GR regulation depends on the binding of auxiliary TFs, other than GR, on the GREs. 56 GREs were enriched for motifs similar to those of the AP-1, ETS/TEAD, and FOX gene families. In another study, computational analysis using ChIP-exo data revealed that FOX factors seem to co-occupy the GREs along with GR, possibly to maintain the open chromatin conformation and allow GR to bind to the genome. 40 The function of AP-1, on the other hand, appears to be limited to providing access to chromatin without directly interacting with the GR on the GREs. 40 In the same study, it was also demonstrated that members of the ETS/TEAD protein families directly interacted with GR and tethered it to the DNA. 40 The regulatory attributes of these elements explain why their motif binding sequences are enriched not only in the GREs of our ChIP-seq sets but in other GRE ChIP-seq studies too. 40,55,57,58 With the intention of identifying potential direct GR targets among the list of DE genes, distinct types of publicly available data were integrated into a custom bioinformatics pipeline, which allowed the ranking of the genes according to how strong the evidence that suggests direct targeting by GR was. The topranked DE genes included well-characterized GR targets such as FKBP5 59 and VIPR1, 33,60 as well as other known islet/b-cell-specific genes such as PDX-1 and NKX6-1, 16 the latter indicating that these results are trustworthy. The top-ranked DE gene in both human islet and EndoC-bH1 samples was ZBTB16, which was recently demonstrated to be induced by dexamethasone in pancreatic islets. 33 The ZBTB16 gene has also demonstrated the highest degree of methylation alterations after dexamethasone treatment in insulin-secreting cells. 23 Previous studies have also linked ZBTB16 with systemic glucose homeostasis as a negative regulator of adipogenesis 61 and insulin sensitivity in skeletal muscle and liver. 62,63 When we investigated the GR binding to ZBTB16 in order to understand the molecular regulation of its function, we found contrary to common belief (regulatory elements reside mostly upstream or downstream of their target genes) that the 10 GREs associated with ZBTB16 were located exclusively on intronic sites of the gene. Nevertheless, intronic GR binding has been recorded before in a relatively high fraction (25-30%) of the total identified GREs, 39,58 with luciferase reporter assay results suggesting a potential functional role of the GBSs of intronic GREs. 64,65 Moreover, while the majority of regions reside in genomic regulatory regions, only 4 GREs in intron 3 and 1 GRE in intron 4 were overlapping with highly conserved regions and contain at least one GBS. The central role of these positions in the glucocorticoid-mediated regulation of ZBTB16 was also supported by the significant induction of open chromatin signal in these regions in human islets, which were treated with both high and low doses of dexamethasone. 33 ChIP followed by PCR in EndoC-bH1 cells revealed a consistent active binding of GR in nine out of the ten predicted GREs, although with some degree of variation between the replicates. The fact that fluctuating levels of islet-specific accessible chromatin signal were also observed in different replicates in control and dexamethasone-treated human islets, 33 indicates a rather dynamic GR regulatory control on ZBTB16 that may depend on GR interaction with TFs that modulate chromatin accessibility. The involvement of auxiliary TFs in the glucocorticoid-GR signaling pathway could also explain the existence of more GR-bound regions without a GBS (six out of ten) than regions with a GBS on the introns of ZBTB16. As at least five out of the ten predicted GR-bound regions on ZBTB16 reside in non-conserved genomic regions (Figure 4, positions: [1][2][3][4]9), it is probable that these regions constitute cell-type specific regulatory elements.
Finally, we disclosed the expression induction of ZBTB16, which characterizes the glucocorticoid effect, on the b-cell function by manipulation of ZBTB16 expression in EndoC-bH1 cells and prediction of direct islet-specific ZBTB16 gene targets with bioinformatics methods. Gene expression assays on EndoC-bH1 showed a significant induction of ZBTB16 expression after dexamethasone treatment that was dependent on the dose and the time under treatment. After incubating EndoC-bH1 cells with dexamethasone, impairment of insulin secretion under high glucose conditions was observed after 48h, which was preceded by decreased cellular insulin content 8h post-treatment. The reduction of insulin content together with the observed increased proliferation rate may suggest that the cells are transitioning to a lower maturity state as has been shown before in c-Myc-expressing INS1 cells. 66 This is supported by the functional annotation of DE genes after dexamethasone treatment where b-cell pancreatic cell differentiation is significantly enriched ( Figure 1C). Moreover, taking into account that ER stress is the top enriched molecular pathway of DE genes after dexamethasone treatment in EndoC cells ( Figure 1C), we hypothesize that reduced insulin content may be a compensatory mechanism of EndoC-bH1 cells against elevated ER stress levels. iScience Article Overexpression of ZBTB16, though, could moderately rescue cellular insulin content and secretion. Moreover, after partially suppressing the dexamethasone-mediated ZBTB16 expression induction, we noticed increased insulin secretion under low glucose conditions, resembling the diabetic phenotype. 68 In the same samples, INS expression was reduced and SGK1 gene induction, which has been associated with insulin release and type 2 diabetes, 24,69 was inhibited. Everything considered, increased expression of ZBTB16 through induction by glucocorticoids may have a protective role hampering the otherwise deleterious effects of glucocorticoids in the b-cell.
Taking one step further, our bioinformatics pipeline also revealed potential direct targets of ZBTB16 in the human islets. Integration of b-cell transcriptome data revealed that 68% of these targets are also expressed in the human pancreatic b-cell. Despite this divergence, pathway analysis uncovered a similar group of significantly enriched terms related to mitochondrial membrane/biogenesis, regulation of translation, and cell cycle/division in both human islet and b-cell targets.
ZBTB16 overexpression has been previously associated with mitochondrial number and function in brown adipocytes. 70 The fact that in fasting conditions ZBTB16 expression is suppressed in b-cells in a similar fashion to the energy-storing white adipose tissue, and is induced in tissues with high-energy requirements such as the brown adipose tissue and skeletal muscle, 62 could imply its implication in stress responses that aim to cover the energy demands of the cell. In EndoC-bH1 cells, while we found dexamethasone treatment negatively affected mitochondrial function, confirming findings from previous studies in insulin-secreting and other cell lines, 71,72 we also found that overexpression of ZBTB16 could completely compensate for these negative effects and restore mitochondrial function to normal levels. As insulin secretion is well known to be coupled to mitochondrial function, these results go in line with the insulin secretion measurements showing that ZBTB16 can restore insulin secretion, reinforcing its protective role in the b-cell. The implication of ZBTB16 in a protective b-cell mechanism via improved mitochondrial function has been shown before in rats undergoing far-infrared radiation. 73 ZBTB16 predicted targets annotated as relevant to mitochondrial membrane and biosynthesis include MED1 74 and TMEM11. 75 Regarding cell cycle control, ZBTB16 has been reported as both negative and positive regulator of cell cycle control in different cell types. [76][77][78][79][80] In our hands, ZBTB16 overexpression or suppression in EndoC-bH did not alter cell viability/proliferation in control conditions and did not have any additive effects on the significantly increased cell proliferation rate upon dexamethasone treatment. In that context, several genes including SPDL1, 81 PDS5A, 82 and SLC12A2 83 were identified as targets that can modulate cell division. It is also worth mentioning that miR-375, a miRNA that is important for b-cell development, proliferation, and secretion 84 was also among the ZBTB16 targets. Among the targets of ZBTB16 there were several genes implicated in the post-transcriptional regulatory mechanism. For instance, ATF4 85 and MAPK14 (alias: p38) 86 both mediate ER function upon cellular stress and AKT2 phosphorylates targets involved in protein synthesis. 87 Taken together, glucocorticoids trigger distinct transcriptome changes in human islets and EndoC-bH1 cells, although with significant similarities. GR binding patterns display considerable similarities between the two and seem to be largely dependent on the interplay with other transcription factors. ZBTB16 is the most highly confident direct GR genetarget in both human islets and EndoC-bH1 cells and its substantial glucocorticoid induction may be able to alleviate the cellular stress by improving mitochondrial function as a part of a compensatory mechanism against the b-cell dysfunction triggered by other glucocorticoid targets. Altogether, this study provides a better insight on the mode of function of glucocorticoids in the pancreatic islets and b-cells, which should be valuable when developing pharmacological treatment strategies against glucocorticoid-induced diabetes in the future.

Limitations of the study
A caveat of the current study is the low number of human islet samples that were used for RNA-sequencing after GC treatment (n = 4 human donors) due to the low availability of such samples. This was the reason that further functional investigation on the ZBTB16 gene was performed only in EndoC-bH1 cells in order to ensure satisfactory number of repeats (n = 4-6 biological replicates/assay). Moreover, we attempted to define GR binding properties in human islets and b-cells by using publicly available ChIP-GR binding sites from other tissues and cell types. Finally, we demonstrated a protective effect of ZBTB16 against the deleterious effects of dexamethasone in EndoC-bH1 cells. However, further investigation is necessary in human islets and in vivo animal models in order to more robustly assess the compensatory beneficial effects of ZBTB16 after glucocorticoid treatment.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  iScience Article iScience Article Western blotting analysis After treating EndoC-bH1 cells with dexamethasone, the cells were lysed and protein concentration was determined with the BCA protein assay kit (Pierce, Rockford, IL, USA). Protein (15 mg homogenate) was separated by 4-15% TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA). Then, the gels were activated with UV light for 1 min to visualize total protein on the blotted LF PVDF membrane (Bio-Rad). The protein was transferred to PVDF membranes, then blocked with 5% milk and 1% BSA in buffer consisting of 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, and 0.1% (v/w) Tween for 1 h. The membranes were individually probed with an antibody against ZBTB16 (1:1000, #39784, Cell Signaling Technology, Danvers, MA, USA). The primary antibody was detected with a horseradish peroxidase conjugated goat anti-rabbit IgG, HRP-linked antibody (1:10 000, #7074, Cell Signaling Technology, Danvers, MA, USA). Protein was detected with Clarity Western ECL Substrate and Bio-Rad ChemiDoc MP Imaging System (Bio-Rad Laboratories) and quantified with Image Lab 6.1 software (Bio-Rad Laboratories), after normalizing the intensity of each protein band to that of the total protein bands in the lane.